CHARACTERIZATION AND ENZYME-CONJUGATION OF A SPECIFIC ANTI-L1 NANOBODY

Abstract

Persistent infection of the human papillomaviruses (HPV) has been shown to result in cervical cancer and intraepithelial neoplasia. Early detection and screening programs are essential strategies against cervical cancer. A nanobody is the smallest antigen-binding fragment known and is derived from a camelid heavy-chain antibody. This tiny protein shows high solubility and stability. It can be produced cost-effectively with high yield production. In this study, we enriched a nanobody library against the L1 protein of HPV. Several colons were selected from this enriched library using monoclonal phage-enzyme linked immunosorbent assay (phage-ELISA) and analyzed for identification of nanobody genes. The expression of nanobody fragments was performed in Rosetta gami2. The C74 nanobody that showed strong binding to the L1 protein of HPV16 was selected, purified, and characterized by Western blotting and ELISA. The selected nanobody was tested for sensitivity, specificity, and affinity. A nanobody conjugated to horseradish peroxidase (HRP) was selected and used for detection of L1 protein of HPV16. This study demonstrates that the C74-HRP, due to its specificity and good binding affinity for a specific viral antigen, is a potential diagnostic tool that can be used as a promising reagent for the new generation of HPV diagnosis approaches.

Keywords:

• cervical cancer
• HPV16
• HRP
• L1 protein
• nanobody

ACKNOWLEDGMENTS

The authors thank Dr. Martin Müller (German Cancer Research Center, Germany) for kindly providing the purified HPV16 L1 protein. This work was partly supported by the Tarbiat Modares University and in part by the University of Isfahan by the office of graduate studies.

Notes

The results represent an average of reactivity (OD₄₅₀) for each round of panning and are presented as mean ± SD. All assays were performed in triplicate.

For selection by Gardasil as antigen in monoclonal phage-ELISA, detection was performed by anti-M13-HRP as secondary antibody and by purified HPV16L1 in ELISA. This was performed by anti-HA-HRP. BSA was used as negative control in both of sets of experiments; in upper row in monoclonal phage-ELISA and in lowest row in ELISA assay. The results represent an average of reactivity (OD₄₅₀) for each clone and are presented as mean ± SD. All assays were performed in triplicate.